Biochemical Basis of T Cell Activation

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Engagement of multicomponent immunoreceptors such as the T cell antigen receptor results in rapid recruitment and activation of multiple protein tyrosine kinases (PTKs) including Lck, Fyn, ZAP-70 and Itk. These PTKs then phosphorylate many enzymes and adapter molecules involved in complex signaling cascades. Our studies have focused on a critical substrate of the PTKs, LAT (linker for activation of T cells), a 36-38kD integral membrane adapter protein. We have performed studies to characterize how LAT is phosphorylated and then binds many critical signaling molecules, thus bringing other adapter molecules and enzymes in multimolecular complexes to the plasma membrane in the vicinity of the activated TCR. Biochemical, biophysical, microscopic and genetic techniques are currently employed to study the characteristics of LAT-based signaling complexes and the enzyme pathways that are coupled to and activated at LAT complexes. Previously we demonstrated that LAT-based structures known as microclusters, as they are visible microscopically, are the site of molecular complex formation occurring with T cell activation. We have used state-of-the-art microscopy including super-resolution analysis to study the molecular organization of these microclusters. We demonstrated that there are two spatially separate domains in the microclusters. One consists of the TCR and associated ZAP-70 PTK and the other contains LAT and LAT-bound signaling molecules. A kinetic analysis of the formation of the microclusters revealed that molecules are recruited in a step-wise fashion beginning with ZAP-70 recruitment to the TCR followed by LAT and other signaling proteins and then by molecules involved in microcluster dissociation. We have also published a super-resolution microscopy study of microclusters. In addition to engagement of the TCR, full T cell activation requires a co-stimulatory signal delivered through the CD28 cell surface receptor. To study the effect of simultaneous stimulation of the TCR and the CD28 molecule we again turned to our microscopic techniques to observe the effect of the CD28 co-stimulatory signal. We showed that CD28 co-stimulation accelerates the recruitment of the ZAP-70 protein tyrosine kinase to the TCR. Additionally, we found that such co-stimulation increases the amount of Lck and, in particular, the amount of activated Lck within the microclusters. This enhanced recruitment of activated Lck would decrease the threshold for activation via the TCR. Multiple protein complexes are found in the microclusters described above. One such protein complex assembles on the LAT adapter molecule. In this protein complex three molecules, the adapters Gads and SLP-76 and the enzyme phospholipase Cgamma1, bind phosphorylated LAT and three more molecules bind SLP-76, the adapter Nck and the enzymes Vav and Itk. Formation of this heptameric complex ensures that phospholipase C-gamma1 reaches the plasma membrane where it can be phosphorylated and thus activated by the protein tyrosine kinase Itk within the complex. Phospholipase C-gamma1 thus activated can cleave phospholipids in the plasma membrane and the consequent products, diacylglycerol and inositol phosphates, proceed to activate additional downstream pathways. The role of Itk in activating the phospholipase has been known for several decades, but it has been impossible to perform a detailed enzymatic study of Itk in the complex because of the inability to purify the enzyme. In our latest study we have now succeeded in purifying the enzyme and performing an initial enzymatic analysis.